Bone density, strength, and formation in adult cathepsin K (-/-) mice.

Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts, that plays a prominent role in degrading Type I collagen. Growing CatK null mice have osteopetrosis associated with a reduced ability to degrade bone matrix. Bone strength and histomorphometric endpoints in young adult CatK null mice aged more than 10 weeks have not been studied. The purpose of this paper is to describe bone mass, strength, resorption, and formation in young adult CatK null mice. In male and female wild-type (WT), heterozygous, and homozygous CatK null mice (total N=50) aged 19 weeks, in-life double fluorochrome labeling was performed. Right femurs and lumbar vertebral bodies 1-3 (LV) were evaluated by dual-energy X-ray absorptiometry (DXA) for bone mineral content (BMC) and bone mineral density (BMD). The trabecular region of the femur and the cortical region of the tibia were evaluated by histomorphometry. The left femur and sixth lumbar vertebral body were tested biomechanically. CatK (-/-) mice show higher BMD at the central and distal femur. Central femur ultimate load was positively influenced by genotype, and was positively correlated with both cortical area and BMC. Lumbar vertebral body ultimate load was also positively correlated to BMC. Genotype did not influence the relationship of ultimate load to BMC in either the central femur or vertebral body. CatK (-/-) mice had less lamellar cortical bone than WT mice. Higher bone volume, trabecular thickness, and trabecular number were observed at the distal femur in CatK (-/-) mice. Smaller marrow cavities were also present at the central femur of CatK (-/-) mice. CatK (-/-) mice exhibited greater trabecular mineralizing surface, associated with normal volume-based formation of trabecular bone. Adult CatK (-/-) mice have higher bone mass in both cortical and cancellous regions than WT mice. Though no direct measures of bone resorption rate were made, the higher cortical bone quantity is associated with a smaller marrow cavity and increased retention of non-lamellar bone, signs of decreased endocortical resorption. The relationship of bone strength to BMC does not differ with genotype, indicating the presence of bone tissue of normal quality in the absence of CatK.

Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity.

Prostaglandin E(2) (PGE(2)) is bone-anabolic, i.e. stimulates bone formation and increases bone mass. In this study, we explored possible intracellular mechanisms of its increase of osteogenic cells in rat bone marrow. Adherent rat bone marrow cells were counted after 12-48 h or cultured for 21 days and mineralized nodules were counted. Also, apoptosis of marrow cells was measured after in vivo PGE(2) injection. PGE(2) (100 nM) increased 2-3 fold the number of adherent BMSC, an effect which was mediated via binding the EP(4) receptor since it was mimicked by forskolin and 11-deoxy-prostaglandin E(1) (PGE(1)) and was blocked by DDA and L-161982 (EP(4) antagonist). PGE(2) stimulated sphingosine kinase (SPK) activity since its effects were blocked by DMS (SPK inhibitor) and mimicked by SPP (SPK product). PGE(2) reduced the activity of caspase-3 and -8 in BMSC and their inhibitors increased BMSC number and nodule formation. In vivo, PGE(2) prevented the increase in the apoptosis of bone marrow cells caused by indomethacin. We propose that PGE(2) exerts an anti-apoptotic effect on BMSC, thereby increasing their number and subsequent osteoblastic differentiation. Such an effect could explain how PGE(2) stimulates bone formation in vivo.

Preservation of thoracic spine microarchitecture by alendronate: comparison of histology and microCT.

The effect of bisphosphonates on trabecular microarchitecture may contribute to the reduced risk of vertebral fracture with treatment independent of the bone volume. Trabecular structure was examined at the twelfth thoracic vertebra after 2 years of treatment of two groups of ovariectomized baboons on high and low doses of alendronate, compared with ovariectomized and non-ovariectomized controls. Standard 2D histological measurements showed that alendronate treatment of ovariectomized animals resulted in significantly higher total trabecular length and a lower marrow star volume in comparison with ovariectomized controls indicating preservation of connectivity. Similarly when the vertebrae were examined using a novel thick slice technique that combines 2D and 3D information, ovariectomy produced a significantly higher number of "real" trabecular termini in comparison with normal. When ovariectomized animals were treated with increasing doses of alendronate, fewer "real" termini were seen. MicroCT analysis (2D and 3D) correlated well with the histological measurements, although more variability and less discrimination between groups was seen, with no statistically significant differences with alendronate treatment. Reduced vertebral fracture risk with alendronate may be due to a combination of factors including the increased bone volume, reduced turnover and greater mineralization reported by others. Added to this is now suggested the preservation of several aspects of vertebral cancellous architecture, with microscopy the most sensitive method of analysis.

Trabecular bone microarchitecture after alendronate treatment of osteoporotic women.

OBJECTIVE: To compare the microarchitecture of iliac crest trabecular bone from women treated for two to three years with alendronate versus that of women treated with placebo. RESEARCH DESIGN AND METHODS: Three-dimensional micro-computed tomography (micro-CT; resolution 20 microm) and two-dimensional histomorphometry (resolution 5-7 microm) were used to examine trabecular bone from single transilial biopsies obtained at the completion of clinical trials. MAIN OUTCOME MEASURES: Microarchitectural variables, including bone volume, trabecular number, trabecular thickness, and trabecular spacing in specimens from alendronate- and placebo-treated women were examined. Three-dimensional images of trabecular bone from both groups were constructed from CT images. Correlations among variables and between techniques were also calculated. RESULTS: Eighty-eight specimens were suitable for evaluation by both techniques. As measured by two-dimensional histomorphometry, bone volume fraction (as a proportion of total volume) and trabecular thickness were significantly greater in alendronate specimens, 17.1 +/- 5.5% vs. 13.4 +/- 5.5% (p = 0.0043) and 127 +/- 29 microm vs. 109 +/- 28 microm (p = 0.0090), respectively, and trabecular spacing was significantly smaller, 729 +/- 227 microm vs. 862 +/- 338 microm (p = 0.005). Micro-CT yielded similar findings: bone volume and trabecular number were significantly greater in alendronate specimens: 19.4 +/- 6.2% vs. 16.2 +/- 6.3% (p = 0.0412) and 1.46(+/-) 0.32 vs. 1.31(+/-) 0.33 per mm (p = 0.0346). Two-dimensional and micro-CT measured characteristics correlated strongly with one another, with Pearson product moment correlation coefficients ranging from 0.60 (for trabecular thickness) to 0.83 (for bone volume). CONCLUSIONS: Trabecular microarchitecture of the ilium, whether studied by two- or three-dimensional methods, is better (greater bone volume, greater trabecular thickness, decreased trabecular spacing) after alendronate treatment than after two to three years of treatment with placebo. Bone volume in a trabecular region is strongly correlated to its microarchitecture, suggesting that bone quantity predicts values for these microarchitectural endpoints.

Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.

Osteoporosis is associated with both atherosclerosis and vascular calcification. No mechanism yet explains the parallel progression of these diseases. Here, we demonstrate that osteoclasts (OCL) depend on lipoproteins to modulate cellular cholesterol levels and that this controls OCL formation and survival. Removal of cholesterol in OCL via high-density lipoprotein or cyclodextrin treatment dose-dependently induced apoptosis, with actin disruption, nuclear condensation and caspase-3 activation. One mechanism linked to the induction of OCL apoptosis was the cell-type-specific failure to induce HMG-CoA reductase mRNA expression, suggesting an absence of feedback regulation of de novo cholesterol biosynthesis. Furthermore, cyclodextrin treatment substantially suppressed essential M-CSF and RANKL-induced survival signaling pathways via Akt, mTOR and S6K. Consistent with these findings, cholesterol delivery via low-density lipoprotein (LDL) significantly increased OCL viability. Interestingly, OCLs from the LDL receptor (LDLR)-/- mouse exhibited reduced size and lifespan in vitro. Remarkably, LDLR+/+ OCL in lipoprotein-deficient medium phenocopied LDLR-/- OCL, while fusion and spreading of LDLR-/- OCL was rescued when cholesterol was chemically delivered during differentiation. With hyperlipidemia being associated with disease of the vascular system and bone, these findings provide novel insights into the selective lipoprotein and cholesterol dependency of the bone resorbing cell. Cell Death and Differentiation (2004) 11, S108-S118. doi:10.1038/sj.cdd.4401399 Published online 12 March 2004

M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase.

Multinucleated bone-resorbing osteoclasts (Ocl) are cells of hematopoietic origin that play a major role in osteoporosis pathophysiology. Ocl survival and activity require M-CSF and RANK ligand (RANKL). M-CSF signals to Akt, while RANKL, like TNFalpha, activates NF-kappaB. We show here that although these are separate pathways in the Ocl, signaling of all three cytokines converges on mammalian target of rapamycin (mTOR) as part of their antiapoptotic action. Accordingly, rapamycin blocks M-CSF- and RANKL-dependent Ocl survival inducing apoptosis, and suppresses in vitro bone resorption proportional to the reduction in Ocl number. The cytokine signaling intermediates for mTOR/ribosomal protein S6 kinase (S6K) activation include phosphatidylinositol-3 kinase, Akt, Erks and geranylgeranylated proteins. Inhibitors of these intermediates suppress cytokine activation of S6K and induce Ocl apoptosis. mTOR regulates protein translation acting via S6K, 4E-BP1 and S6. We find that inhibition of translation by other mechanisms also induces Ocl apoptosis, demonstrating that Ocl survival is highly sensitive to continuous de novo protein synthesis. This study thus identifies mTOR/S6K as an essential signaling pathway engaged in the stimulation of cell survival in osteoclasts.

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis.

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.

Alendronate increases degree and uniformity of mineralization in cancellous bone and decreases the porosity in cortical bone of osteoporotic women.

The strength of bone is correlated with bone mass but is also influenced significantly by other factors such as structural properties of the matrix (e.g., collagen mutations) and the mineral. Changes at all levels of this organization could contribute to fracture risk. We investigated the effects of alendronate (Aln) treatment on the density of mineralization and the ultrastructure of the mineral/collagen composite, size and habitus of mineral particles in iliac cancellous bone, as well as on the porosity of iliac cortical bone from postmenopausal osteoporotic women. Twenty-four transiliac bone biopsies from Phase III Aln (10 mg/day) trials (placebo and Aln after 2 and 3 years of treatment, n = 6 per group) were studied. The mineral structure was investigated by quantitative backscattered electron imaging (qBEI) and by scanning small-angle X-ray scattering (scanning-SAXS). qBEI histograms reflect the bone mineralization density distribution (BMDD), whereas SAXS patterns characterize the size and arrangement of the mineral particles in bone. We found that: (i) the relative calcium content of osteoporotic bone was significantly lower than that of data-base controls; (ii) mineralization was significantly higher and more uniform after Aln treatment; (iii) size and habitus of the mineral particles was not different between placebo and Aln-treated groups; and (iv) the porosity of cortical bone was reduced significantly by Aln treatment. We conclude that Aln treatment increases the degree and uniformity of bone matrix mineralization without affecting the size and habitus of the mineral crystals. It also decreases the porosity of the corticalis. Together these effects may contribute to the observed reduction in fractures.

Prostaglandin receptor EP(4) mediates the bone anabolic effects of PGE(2).

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts.

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

Expression of the prostaglandin E(2) (PGE(2)) receptor subtype EP(4) and its regulation by PGE(2) in osteoblastic cell lines and adult rat bone tissue.

Prostaglandins E (especially PGE(2)) stimulate bone formation and increase bone mass in several species including man. The mechanism for this effect, the target cells, and the receptors involved are not known. Specific cell-surface receptors for PGE(2) (EP(1-4)) have been cloned and characterized. EP(4) was reported to be the major receptor in embryonic and neonatal bone tissue in mice, especially in preosteoblasts; however, no data are available regarding its expression in adult bone. This study examines the expression of EP(4) in bone tissue of young adult rats, in which PGE(2) is markedly anabolic, and in various osteoblastic cell lines. Using northern blot analysis, we found that osteoblastic cell lines RCT-1, RCT-3, TRAB-11, and RP-1, primary osteoblastic cells harvested from fetal rat calvaria, as well as tibiae and calvariae of 5-week-old rats express 3.8 kb EP(4) messenger RNA (mRNA). Treatment of periosteal cells (RP-1) in vitro with 10(-6) mol/L PGE(2) increased the levels of both EP(4) mRNA and EP(4) protein, peaking at 1-2 h. Similarly, systemic administration of an anabolic dose of PGE(2) (3-6 mg/kg) to young adult rats upregulated the expression of EP(4) in the tibia and calvaria, also peaking at 1-2 h. Using in situ hybridization, we found increased expression of EP(4) in bone marrow cells of the tibial metaphysis in response to systemic PGE(2) treatment. The preosteoblastic nature of these EP(4)-expressing cells was suggested by the fact that dexamethasone-treated bone marrow stromal cells in culture express EP(4) mRNA, which is upregulated by PGE(2). Northern blot analysis failed to detect both basal and PGE(2)-induced EP(2) mRNA in the bone samples or cell lines tested. Taken together, these data implicate EP(4) as the major cyclic AMP-related PGE(2) receptor subtype expressed in bone tissue and osteoblastic cells and indicate that this receptor is upregulated by its ligand, PGE(2).

Nitrogen-bisphosphonates block retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation.

The surprising discovery that nitrogen-containing bisphosphonates (N-BPs) act via inhibition of the mevalonate-to-cholesterol pathway raised the possibility that esophageal irritation by N-BPs is mechanism-based. We used normal human epidermal keratinocytes (NHEKs) to model N-BP effects on stratified squamous epithelium of the esophagus. The N-BPs alendronate and risedronate inhibited NHEK growth in a dose-dependent manner without inducing apoptosis. N-BPs (30 microM) caused accumulation of cells in S phase and increased binucleation (inhibited cytokinesis). Consistent with N-BP inhibition of isoprenylation, geranylgeraniol or farnesol prevented accumulation in S phase. Binucleation was also induced by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and by the squalene synthase inhibitor zaragozic acid A and was prevented by adding low-density lipoprotein. At 300 microM, N-BPs reduced expression of cyclin-dependent kinase (cdk) 2 and cdk4 and enhanced expression of p21(waf1) and p27(kip1) and their binding to cdks with corollary hypophosphorylation of retinoblastoma. Lovastatin and zaragozic acid A produced similar effects, except that p21(waf1) expression and binding to cdks was not induced. Growth inhibition, but not binucleation, was also caused by the geranylgeranyl transferase I inhibitor, GGTI-298, which also enhanced cdk2 and cdk4 association with p27(kip1). These findings are consistent with suppression of epithelial cell growth by N-BPs via inhibition of the mevalonate pathway and the consequent reduction in cholesterol synthesis, which blocks cytokinesis, and in geranylgeranylation, which interferes with progression through the cell cycle.

Inhibition of osteoclast function by adenovirus expressing antisense protein-tyrosine kinase 2.

Osteoclast activation is initiated by adhesion to bone, cytoskeletal rearrangement, formation of the sealing zone, and formation of the polarized ruffled membrane. Previous findings suggest that protein-tyrosine kinase 2 (PYK2), a cytoplasmic kinase related to focal adhesion kinase, participates in these events. This study examines the role of PYK2 in adhesion-mediated signaling and osteoclast function, using PYK2 antisense. We produced a recombinant adenovirus containing a 300-base pair reversed 5'-coding region of PYK2 and used full-length PYK2 as a control. Murine osteoclast-like cells or their mononuclear precursors were generated in a co-culture of bone marrow and osteoblasts. Infection with antisense adenovirus significantly reduced the expression of endogenous PYK2 protein relative to uninfected cells or to cells infected with sense PYK2 and caused: 1) a reduction in osteoclast formation in vitro; 2) inhibition of cell spreading and of actin ring formation in osteoclasts plated on glass or bone and of attachment and spreading of osteoclast precursors plated on vitronectin; 3) inhibition of bone resorption in vitro; 4) marked reduction in p130(Cas) tyrosine phosphorylation; and 5) no change in alpha(v)beta(3) integrin expression or c-Src tyrosine phosphorylation. Taken together, these findings support the hypothesis that PYK2 plays a central role in the adhesion-dependent cytoskeletal organization and sealing zone formation required for osteoclastic bone resorption.

Abnormal localisation and hyperclustering of (alpha)(V)(beta)(3) integrins and associated proteins in Src-deficient or tyrphostin A9-treated osteoclasts.

The non-receptor tyrosine kinase Src was shown to be essential for osteoclast function in vivo. We have previously reported that engagement of (alpha)(v)(beta)(3) integrin in osteoclasts induces tyrosine phosphorylation and activation of the adhesion kinase PYK2 and the adaptor protein p130(Cas) in a Src-dependent manner. The objective of this study was to analyse the role of c-Src in the (alpha)(v)(beta)(3) integrin-dependent recruitment of signalling and cytoskeletal molecules in osteoclasts during bone resorption. Using prefusion osteoclasts (pOCs) obtained from cocultures of osteoblasts and spleen cells isolated from Src(-/-) mice or their normal littermates, we found: (1) similar expression levels and ligand binding affinities of (alpha)(v)(beta)(3) integrins in Src(-/-) and Src(+/?) pOCs, (2) reduced adhesion and spreading of Src(-/-) pOCs, (3) defective organisation of the microfilament proteins, F-actin, vinculin and paxillin, and of PYK2 and p130(Cas) in the sealing zone of Src(-/-)OCLs, and (4) hyperclustering of (alpha)(v)(beta)(3) integrins together with microfilament and signalling proteins in the basal membrane of Src-deficient OCLs. In normal OCLs, the tyrosine kinase inhibitor tyrphostin A9 inhibits actin ring formation, bone resorption and tyrosine phosphorylation of several proteins, including c-Src. Furthermore, tyrphostin A9 induced similar hyperclustering of (alpha)(v)(beta)(3) integrins in osteoclasts as observed in Src(-/-) OCLs. Taken together, these findings suggest that normal localisation of (alpha)(v)(beta)(3) and recruitment of its downstream effectors to the appropriate compartments of the osteoclast during resorption depend on Src kinase activity.

In vivo effects of bisphosphonates on the osteoclast mevalonate pathway.

Estrogen deficiency is a leading cause of osteoporosis associated with increased osteoclastic bone resorption. In vitro studies indicate that the clinically used nitrogen-containing bisphosphonates (N-BPs) such as alendronate (ALN), risedronate (RIS) and ibandronate (IBA) suppress bone resorption via inhibition of the mevalonate pathway enzyme farnesyl diphosphate (FPP) synthase in osteoclasts (Ocs). The object of this study was to test the hypothesis that N-BPs inhibit the mevalonate pathway of Ocs in vivo. The mevalonate pathway enzyme hydroxymethyl-glutaryl-coenzyme A reductase (HMGR), is modulated by feedback inhibition from downstream metabolites. We therefore evaluated the in vivo expression of HMGR in Ocs from animals treated with BP. The N-BPs, ALN, IBA and RIS, selectively suppressed HMGR expression in up to 85% of rat tibia osteoclasts, after 48 hr treatment. Etidronate and clodronate, bisphosphonates that do not inhibit FPP synthase, were without effect. Simvastatin treatment opposed ALN reduction of HMGR expression, suggesting regulation by a metabolite(s) between mevalonate and FPP. These data provide the first in vivo evidence for N-BP effects on the mevalonate pathway in osteoclasts, and strongly support the hypothesis that N-BPs act via this mechanism.

PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation.

Pyk2 is a member of the focal adhesion kinase (FAK) family, highly expressed in the central nervous system and haemopoietic cells. Although Pyk2 is homologous to FAK, its role in signaling pathways was shown to be distinct from that of FAK. We show here that Pyk2 is highly expressed in peritoneal IC-21 macrophage and is tyrosine phosphorylated in response to cell attachment to fibronectin and fibrinogen. Upon IC-21 cell adhesion, Pyk2 tyrosine phosphorylation is inhibited by blocking antibodies to the integrin subunits alpha(M) and beta(2). Furthermore, Pyk2 is rapidly tyrosine phosphorylated in response to ligation of beta(2) integrins by antibodies. In migrating macrophages, Pyk2 localizes to perinuclear regions and to podosomes, where it is clustered with tyrosine phosphorylated proteins. Furthermore, in the podosomal ring structure, which surrounds the central actin core, Pyk2 co-localizes with vinculin, talin, and paxillin. In the podosomes, Pyk2 also co-localizes with the integrin alpha(M)beta(2). Lastly, reduction of Pyk2 expression in macrophages leads to inhibition of cell migration. We propose that Pyk2 is functionally linked to the formation of podosomes where it mediates the integrin-cytoskeleton interface and regulates cell spreading and migration.

Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist.

Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.

Therapeutic approaches to bone diseases.

The strength and integrity of our bones depends on maintaining a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. As we age or as a result of disease, this delicate balancing act becomes tipped in favor of osteoclasts so that bone resorption exceeds bone formation, rendering bones brittle and prone to fracture. A better understanding of the biology of osteoclasts and osteoblasts is providing opportunities for developing therapeutics to treat diseases of bone. Drugs that inhibit the formation or activity of osteoclasts are valuable for treating osteoporosis, Paget's disease, and inflammation of bone associated with rheumatoid arthritis or periodontal disease. Far less attention has been paid to promoting bone formation with, for example, growth factors or hormones, an approach that would be a valuable adjunct therapy for patients receiving inhibitors of bone resorption.

Integrins and signaling in osteoclast function.

Integrins are heterodimeric adhesion receptors that mediate cell-matrix and cell-cell interactions. Osteoclasts highly express the alphavbeta3 integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin and bone sialoprotein. RGD-containing peptides, RGD-mimetics and alphavbeta3 blocking antibodies inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in osteoclast function. RGD-containing peptides were shown to raise cytosolic calcium in osteoclasts. Furthermore, several signaling and adaptor molecules were found to be involved in alphavbeta3 integrin-dependent signaling pathways, including phosphatidylinositol 3-kinase, c-Src, PYK2 and p130(cas). In addition, cytoskeletal molecules such as paxillin, vinculin, gelsolin and F-actin are recruited to adhesion contacts upon integrin activation. Many of these molecules signaling and cytoskeletal localize to the sealing zone of actively resorbing osteoclasts, suggesting that they play a role in linking the adhesion of osteoclasts to the bone matrix with the cytoskeletal organization and the polarization and activation of these cells for bone resorption.